Abstract

Clostridium difficile is the leading cause of infectious diarrhoea in hospitals worldwide, because of its virulence, spore-forming ability and persistence. C. difficile-associated diseases are induced by antibiotic treatment or disruption of the normal gastrointestinal flora. Recently, morbidity and mortality resulting from C. difficile-associated diseases have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns. Since 2002, epidemic toxinotype III NAP1/027 strains, which produce high levels of the major virulence factors, toxin A and toxin B, have emerged. These toxins have 63% amino acid sequence similarity and are members of the large clostridial glucosylating toxin family, which are monoglucosyltransferases that are pro-inflammatory, cytotoxic and enterotoxic in the human colon. Inside host cells, both toxins catalyse the transfer of glucose onto the Rho family of GTPases, leading to cell death. However, the role of these toxins in the context of a C. difficile infection is unknown. Here we describe the construction of isogenic tcdA and tcdB (encoding toxin A and B, respectively) mutants of a virulent C. difficile strain and their use in the hamster disease model to show that toxin B is a key virulence determinant. Previous studies showed that purified toxin A alone can induce most of the pathology observed after infection of hamsters with C. difficile and that toxin B is not toxic in animals unless it is co-administered with toxin A, suggesting that the toxins act synergistically. Our work provides evidence that toxin B, not toxin A, is essential for virulence. Furthermore, it is clear that the importance of these toxins in the context of infection cannot be predicted exclusively from studies using purified toxins, reinforcing the importance of using the natural infection process to dissect the role of toxins in disease.

Genetic organization of the C. difficile PaLoc and analysis of toxin mutants. (a) PaLoc genetic organization. XbaI sites (X) are shown, with the PaLoc co-ordinate indicated. Boxes indicate regions used in the recombination vectors and the tcdB- and tcdA-specific probes. Ovals show the gene-specific promoters and arrows the direction of transcription. (b) Structural organization of toxin B (toxin A is similar). Co-ordinates indicate the amino acid number. Relevant domains are shown. The box indicates the region in the recombination vectors. (c) Southern hybridization of mutants. Genomic DNA was XbaI-digested and blots probed as shown. λ-HindIII markers are indicated (kb). WT represents strain JIR8094, the mutant lanes are indicated. DNA was purified from cultures grown in the absence (−) or presence (+) of thiamphenicol selection.

Comparative analysis of toxin production by wild-type and mutants. The wild-type is JIR8094 and the avirulent strain is CD37, which does not produce toxins. (a) Western blot using toxin A-specific antibodies. (b) and (d) Toxin A and toxin B cytotoxicity assays using HT-29 and Vero cells, respectively. Data represent the mean±SEM. n=3. (c) and (e) Neutralization of toxicity. Culture supernatants were added to the cells and morphological changes observed after 24 h (No Ab) or pretreated with toxin A (anti-TcdA) and toxin B (anti-TcdB) neutralizing antibodies prior to their addition to the cells. Representative images are shown. No neutralization was observed when supernatants were pretreated with anti-ErmB methylase antibodies (data not shown). Bar indicates 1 mm.

Virulence of C. difficile wild-type and mutant strains in hamsters. (a) Days from inoculation of C. difficile to colonization of clindamycin treated hamsters. The rate of colonization was 10/10 for WT, 9/10 for tcdAΩ1, 8/10 for tcdAΩ2, 9/10 for tcdBΩ1 and 9/10 for tcdBΩ2. n=10. (b) Days from colonization to death or sacrifice. All hamsters that survived were sacrificed at day 30. n=10. (c) Kaplan-Meier survival estimate demonstrating days from colonization with C. difficile strains to death. n=10.